Vaccination using plasmid DNA has the potential to provide enhanced safety and efficacy over conventional vaccines with increased stability, and accelerated product development. Unfortunately, vaccination of primates with DNA requires multiple inoculations using excessively large doses of plasmid. The A1 domains of cholera toxin (CTA1) and the related heat-labile enterotoxin (LTA1) have demonstrated particular promise as adjuvants that can enhance the immunogenicity of DNA vaccines in small animals and may provide a necessary dose sparing effect in primates. To maximally exploit this promise, however, the activity of CTA1 or LTA1 in vivo should be improved. Our objective is to identify modifications to CTA1 or LTA1 that will enhance their activity in [unreadable] vivo by pursuing the following aims: Aim 1: Identify mutations in the active site of either CTA1 or LTA1 that enhance enzymatic activity in vitro; Aim 2: Identify fusion constructs between CTA1 or LTA1 and human ARF6 that enhance enzymatic activity in vitro; and, Aim 3: Identify constructs from Aims 1, and 2 that optimally enhance the immunogenicity of a candidate SIMmac239-gag DNA vaccine in vivo. [unreadable] Candidate constructs that display a minimum of 10-fold enhanced adjuvanticity in vivo will be selected for evaluation in primate studies to be proposed in phase 2. Successful candidates in phase 2 will be incorporated into candidate HIV DNA vaccines intended for clinical evaluation. [unreadable] [unreadable] [unreadable]